NanoBRET® TE POLQ Helicase and Polymerase Domain Assays
Measure Intracellular Compound Affinity and Occupancy for Helicase or Polymerase Domains of Polymerase Theta
- Quantify compound affinity and fractional occupancy at either helicase or polymerase domains for full-length Polymerase Theta in live cells
- Evaluate target engagement to the polymerase domain at Polymerase Theta/DNA complexes
- Optimized reagents with a simple, addition-only workflow
Catalog Number:
Choose the POLQ domain
Choose an option
Catalog Number: CS3489A04
Catalog Number: CS3489A23
Catalog Number: CS3489A33
Catalog Number: CS3489A32
Catalog Number: CS3489A05
Catalog Number: CS3489A24
Measure Target Engagement to Polymerase Theta in Live Cells
Polymerase Theta (Pol-theta), encoded by the POLQ gene, is the predominant mediator for the Theta-Mediated End-Joining (TMEJ) pathway, one of four pathways of DNA double-strand break repair. Pol-theta has emerged as a therapeutic target in homologous recombination-deficient cancer cells, leveraging the synthetic lethal dependency on alternative DNA repair pathways exhibited by these cells.
The NanoBRET® TE POLQ Helicase and Polymerase Domain Assays can be used to quantify compound affinity and fractional occupancy against Pol-theta in live cells. The assays are based on the NanoBRET® technology, an energy transfer technique designed to measure molecular proximity in living cells.
Want to accelerate your drug discovery workflow? Our Tailored R&D Solutions team offers services using the NanoBRET® TE Assays for Pol-theta.
How the NanoBRET® TE POLQ Helicase and Polymerase Domain Assays Work
NanoBRET® TE Assays quantify target engagement using bioluminescence resonance energy transfer (BRET). These BRET-based assays rely on the transfer of luminescent energy from a NanoLuc® luciferase-target fusion protein expressed in cells to a cell-permeable fluorescent NanoBRET® tracer that binds to the target protein.
In the NanoBRET® TE POLQ Helicase and Polymerase Domain Assays, Pol-theta is expressed as a NanoLuc® luciferase fusion in mammalian cells. To generate a BRET signal, a fixed concentration of a cell-permeable fluorescent NanoBRET® TE tracer is added to these cells and binds to the NanoLuc®-Pol-theta fusion protein. Addition of compounds that bind to Pol-theta results in a dose-dependent decrease in BRET signal. This data allows quantitation of the intracellular affinity of compounds for Pol-theta in live cells.
Assess Domain-Specific Target Engagement
Pol-theta is a large protein with two distinct functional domains (polymerase and helicase) that are separated by an unstructured region. Each domain of Pol-theta has proven vulnerable to small-molecule inhibitors.
To assess target engagement at each domain in full-length Pol-theta, two domain-selective NanoBRET® TE POLQ assays are available. The NanoBRET® TE POLQ Polymerase Domain Assay uses a polymerase domain-specific tracer along with the POLQ-NanoLuc® Fusion Vector that places NanoLuc® luciferase in close proximity to the polymerase domain. A helicase domain-selective tracer, together with the NanoLuc®-POLQ Fusion Vector, are used in the NanoBRET® TE POLQ Helicase Domain Assay.
Evaluate Target Engagement to the Polymerase Domain at Pol-theta/DNA Complexes
Compound engagement to the polymerase domain of Pol-theta has shown cooperativity with DNA binding. To enable the measurement of compound binding to the polymerase domain at the nucleoprotein complex, we developed an optimized workflow for the NanoBRET® TE POLQ Polymerase Domain Assay to introduce small DNA fragments into live cells, mimicking the presence of damaged DNA. The presence of the DNA fragments potentiated the BRET signal from the polymerase domain tracer and the affinity of the unlabeled test compound. These results support the known mechanisms of uncompetitive target engagement, with respect to DNA binding, of the Pol-theta polymerase domain inhibitors.
Compound affinity measured in the NanoBRET® TE Assay correlates with the published potency derived from a TMEJ reporter assay. See our Scientific Poster for more details.
Assess Domain Specificity of Pol-Theta Inhibitors Targeting the Helicase Domain
The NanoBRET® TE POLQ Helicase Domain Assay incorporates a separate NanoBRET® Tracer directed to the helicase domain of Pol-theta and a NanoLuc®-POLQ fusion vector that places NanoLuc® luciferase near the helicase domain. This system enables measurement of domain specificity of Pol-theta inhibitors. Tracer displacement is observed with helicase domain inhibitors, while known polymerase domain inhibitors do not demonstrate target engagement.
What You Need to Perform the NanoBRET® TE Assay
POLQ Polymerase Domain Assay
We supply:
- NanoBRET® Tracer POLQ Polymerase*
- POLQ-NanoLuc® Fusion Vector (Pol site)
or POLQ-NanoLuc® HEK293 Stable Cell Line (Pol site) - Damaged DNA Mimic*
Additional reagents needed for performing the NanoBRET® TE Assay are listed below. A full list of required materials can be found in the Application Note for the assay.
POLQ Helicase Domain Assay
We supply:
- NanoBRET® Tracer POLQ Helicase*
- NanoLuc®-POLQ Fusion Vector (Heli site)
Additional reagents needed for performing the NanoBRET® TE Assay are listed below. A full list of required materials can be found in the Application Note for the assay.
*Each vial is sufficient for at least 1,000 assays when used as recommended in the Application Note.
Patents and Disclaimers
Materials may be covered by pending or issued patents or may have certain limitations.
Information is available upon request and will be included on applicable quotes.
Specifications
Catalog Number:
Promega DNA Vector Limited Use Label License (LULL)
BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.
-
1. Usage Restrictions:
- 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation.
- 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including but not limited to product manufacture or resale of the material for any use and sale of derivatives for any use.
- 1.3. If recipient is a contract research organization (“CRO”), recipient may transfer or sell data or information generated using the material or derivatives to CRO’s customers who request such data or information, provided that the material or derivatives cannot be transferred from CRO to CRO’s customers.
-
2. Modification Restrictions:
- 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors.
- 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes.
-
3. For determinations of luminescence activity of this material and its derivatives, recipient must either:
- 3.1. Use Promega-branded luminescent assay reagents (LARs); or
- 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
-
4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either:
- 4.1. Use Nano-Glo®-branded LARs; or
- 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
-
5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either:
- 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or
- 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
-
6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must:
- 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and
- 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or
- 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein.
-
7. For uses of HaloTag® Technology in this material, recipient must either:
- 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or
- 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.
-
8. Transfer of Materials:
- 8.1. Unmodified material or progeny of such material cannot be transferred.
- 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license.
- 8.3. If recipient is not a CRO, user may transfer this material to a CRO in order to have CRO use the material solely on behalf of recipient to provide recipient data or information, and CRO may transfer or sell such data or information to recipient, provided that recipient provides CRO with a copy of this label license and CRO agrees to comply with this label license.
- 8.4. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party.
-
9. Other Uses:
- 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information.
-
10. Disclaimer and Governing Law:
- 10.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
NanoBRET™ TE Tracers V2 Limited Use Label License
BY USE OF THIS MATERIAL, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.
Researcher may use this material for research use only; no commercial use is allowed. Commercial use means any and all uses of this material by a party in exchange for consideration, including, but not limited to
- (1) use in further product manufacture;
- (2) use in provision of services, information or data; and
- (3) resale of the material, whether or not such material is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the material. No other use or transfer of this material is authorized without the prior express written consent of Promega.
For uses of this product for energy transfer (such as bioluminescence resonance energy transfer), researcher must:
- (a) use NanoLuc®-branded luciferase or derivatives, or subunits of the NanoBiT® technology for all energy transfer determinations conducted with this material; and
- (b) use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor, or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity; or
- (c) contact Promega to obtain a license for use of the material with reagents not listed above or manufactured by Promega.
In addition, researcher must:
Contact Promega to obtain a license for use of the material in assays not involving bioluminescence resonance energy transfer (BRET).
With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
Limited Use Label License
BY USE OF THIS MATERIAL, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.
Researcher may use this material for research use only; no transfer, or commercial use of this material is allowed. Commercial use means any and all uses of this material by a party in exchange for consideration, including, but not limited to
- (1) use in further material manufacture; and
- (2) resale of the material, whether or not such material is resold for use in research. Researcher shall have no right to modify or otherwise create variations or derivatives of the material. No other use of this material is authorized without the prior express written consent of Promega. Notwithstanding the foregoing, researcher may use this material in provision of services, information or data to third parties in exchange for consideration, provided that researcher does not transfer the material. No other use of this material is authorized without the prior express written consent of Promega.
With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
Promega Cell Line Limited Use Label License (LULL)
BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.
-
1. Usage and Transfer Restrictions:
- 1.1. Recipient may use this material for research use only, which includes but is not limited to drug discovery and development, quality assurance testing and product release assays. No transfer, resale or commercial use of this material is allowed without the written consent of Promega Corporation.
- 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including product manufacture, providing a service, information or data to unaffiliated third parties, resale of the material for any use and sale of derivatives for any use.
-
2. Modification Restrictions:
- 2.1. Recipient shall have no right to modify, derivatize or otherwise create variations of the nucleotide sequence of any proprietary gene, including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes stably transfected within the cells.
- 2.2. Recipient may genetically engineer the cell line using exogenous nucleic acid sequences of the recipient’s choosing.
-
3. Propagation and Storage:
- 3.1. The recipient may not propagate the cells, unless material is marked as Propagation Model, Cell Bank or Master Cell Bank; then recipient may propagate and store the cells for long-term use.
-
4. For all determinations of luminescence or other assay-related activity of this material and its derivatives, recipient must either:
- 4.1. Use the detection systems specified in the protocol or data report and purchased from Promega for all luminescence assays when using this material; or
- 4.2. Contact Promega to obtain a license for use of this material with reagents other than a Bio-Glo™ branded luciferase reagent, including but not limited to:
- 4.2.1 Bio-Glo™, Bio-Glo-NL™, Bio-Glo-NB™ TCK, Bio-Glo-NB™ VLP, Bio-Glo-NB™ Lytic
-
5. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must:
- 5.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and
- 5.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or
- 5.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein.
-
6. For uses of HaloTag® Technology in this material, recipient must either:
- 6.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or
- 6.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.
-
7. Disclaimer and Governing Law:
- 7.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
Promega DNA Vector Limited Use Label License (LULL)
BY USE OF THIS MATERIAL, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.
-
1. Usage Restrictions:
- 1.1. Recipient may use this material for research use only; no commercial use of this material is allowed without the written consent of Promega Corporation.
- 1.2. “Commercial use” means any and all uses of this material or derivatives by recipient for monetary or other consideration, including but not limited to product manufacture or resale of the material for any use and sale of derivatives for any use.
- 1.3. If recipient is a contract research organization (“CRO”), recipient may transfer or sell data or information generated using the material or derivatives to CRO’s customers who request such data or information, provided that the material or derivatives cannot be transferred from CRO to CRO’s customers.
-
2. Modification Restrictions:
- 2.1. Recipient shall have no right to modify or otherwise create variations of the nucleotide sequence of any proprietary gene including but not limited to luciferase, NanoBiT® technology (e.g., HiBiT), HaloTag® technology or genes included in fusion vectors.
- 2.2. Recipient shall have the right to make derivatives of cloning vectors by incorporating exogenous sequences provided the resulting fusion gene has <4 nucleotides deleted from the affected terminus of the proprietary genes.
-
3. For determinations of luminescence activity of this material and its derivatives, recipient must either:
- 3.1. Use Promega-branded luminescent assay reagents (LARs); or
- 3.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
-
4. For determinations of NanoLuc® or NanoBiT® luminescence activity of this material and its derivatives, recipient must either:
- 4.1. Use Nano-Glo®-branded LARs; or
- 4.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
-
5. For determinations of GloSensor™ activity of this material and its derivatives, recipient must either:
- 5.1. Use GloSensor™ cAMP Reagent for in vitro and in-cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives); or
- 5.2. Contact Promega to obtain a license for the use of LARs not manufactured by Promega.
-
6. For uses of this material for energy transfer (such as bioluminescence resonance energy transfer), recipient must:
- 6.1. Use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity by this material and its derivatives; and
- 6.2. Use NanoBRET™-branded energy acceptors (e.g., NanoBRET™ tracers, NanoBRET™ dyes, BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this material and its derivatives; or
- 6.3. Contact Promega to obtain a license for the use of the material and its derivatives for energy transfer assays to energy acceptors not manufactured by Promega. No license is needed if the energy transfer acceptor is a genetically encoded auto-fluorescent protein.
-
7. For uses of HaloTag® Technology in this material, recipient must either:
- 7.1. Use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties; or
- 7.2. Contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.
-
8. Transfer of Materials:
- 8.1. Unmodified material or progeny of such material cannot be transferred.
- 8.2. Derivatives of cloning vectors can be transferred to others for research use provided that at the time of transfer a copy of this label license is given to the recipients and recipients agree to be bound by the terms of this label license.
- 8.3. If recipient is not a CRO, user may transfer this material to a CRO in order to have CRO use the material solely on behalf of recipient to provide recipient data or information, and CRO may transfer or sell such data or information to recipient, provided that recipient provides CRO with a copy of this label license and CRO agrees to comply with this label license.
- 8.4. Notwithstanding any other provision in this label license, the recipient can transfer the material (original or derivative of a cloning vector) to a third party for the sole purpose of amplification of the vector, provided that at the time of transfer a copy of this label license is given to the third party and the third party agrees to be bound by the terms of this label license. The third party may not use any of the material (original or amplified) for its own purposes or further transfer the material to any other party.
-
9. Other Uses:
- 9.1. For any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, contact Promega for supply and licensing information.
-
10. Disclaimer and Governing Law:
- 10.1. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
NanoBRET™ TE Tracers V2 Limited Use Label License
BY USE OF THIS MATERIAL, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE.
Researcher may use this material for research use only; no commercial use is allowed. Commercial use means any and all uses of this material by a party in exchange for consideration, including, but not limited to
- (1) use in further product manufacture;
- (2) use in provision of services, information or data; and
- (3) resale of the material, whether or not such material is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the material. No other use or transfer of this material is authorized without the prior express written consent of Promega.
For uses of this product for energy transfer (such as bioluminescence resonance energy transfer), researcher must:
- (a) use NanoLuc®-branded luciferase or derivatives, or subunits of the NanoBiT® technology for all energy transfer determinations conducted with this material; and
- (b) use NanoBRET™-branded luminescent assay reagents (LARs; e.g., NanoBRET™ Nano-Glo® Substrate), Intracellular TE Nano-Glo® Substrate/Inhibitor, or Intracellular TE Nano-Glo® Vivazine™/Inhibitor for all determinations of luminescent activity; or
- (c) contact Promega to obtain a license for use of the material with reagents not listed above or manufactured by Promega.
In addition, researcher must:
Contact Promega to obtain a license for use of the material in assays not involving bioluminescence resonance energy transfer (BRET).
With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE MATERIAL. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
Resources
Featured Resource
A NanoBRET® Target Engagement Assay for querying domain selectivity at full-length Polymerase Theta in live cells
Two domain-selective, live-cell NanoBRET® Target Engagement (TE) Assays were developed for full-length Polymerase Theta, providing simple workflows for quantifying compound binding to either helicase or polymerase domains. Small DNA fragments are introduced into live cells when performing the polymerase domain TE assay, which enables the measurement of compound engagement at Polymerase-Theta/DNA complex. These two assays allow mechanism-of-action studies at Polymerase-Theta in living cells.
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